High-Performance Molecular Dynamics Simulations for Native Mass Spectrometry of Large Protein Complexes with the Fast Multipole Method.

Native mass spectrometry (MS) is widely employed to study the structures and assemblies of proteins ranging from small monomers to megadalton complexes. Molecular dynamics (MD) simulation is a useful complement as it provides the spatial detail that native MS cannot offer. However, MD simulations performed in the gas phase have suffered from rapidly increasing computational costs with the system size.

A druggable conformational switch in the c-MYC transactivation domain.

The c-MYC oncogene is activated in over 70% of all human cancers. The intrinsic disorder of the c-MYC transcription factor facilitates molecular interactions that regulate numerous biological pathways, but severely limits efforts to target its function for cancer therapy. Here, we use a reductionist strategy to characterize the dynamic and structural heterogeneity of the c-MYC protein.

Advances in mass spectrometry to unravel the structure and function of protein condensates.

Membrane-less organelles assemble through liquid-liquid phase separation (LLPS) of partially disordered proteins into highly specialized microenvironments. Currently, it is challenging to obtain a clear understanding of the relationship between the structure and function of phase-separated protein assemblies, owing to their size, dynamics and heterogeneity. In this Perspective, we discuss recent advances in mass spectrometry (MS) that offer several promising approaches for the study of protein LLPS.

Mass Spectrometry of RNA-Binding Proteins during Liquid-Liquid Phase Separation Reveals Distinct Assembly Mechanisms and Droplet Architectures.

Liquid-liquid phase separation (LLPS) of heterogeneous ribonucleoproteins (hnRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility mass spectrometry, and molecular dynamics simulations. We used an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage.

Liquid-Liquid Phase Separation Primes Spider Silk Proteins for Fiber Formation via a Conditional Sticker Domain.

Many protein condensates can convert to fibrillar aggregates, but the underlying mechanisms are unclear. Liquid-liquid phase separation (LLPS) of spider silk proteins, spidroins, suggests a regulatory switch between both states. Here, we combine microscopy and native mass spectrometry to investigate the influence of protein sequence, ions, and regulatory domains on spidroin LLPS.

A "grappling hook" interaction connects self-assembly and chaperone activity of Nucleophosmin 1.

How the self-assembly of partially disordered proteins generates functional compartments in the cytoplasm and particularly in the nucleus is poorly understood. Nucleophosmin 1 (NPM1) is an abundant nucleolar protein that forms large oligomers and undergoes liquid-liquid phase separation by binding RNA or ribosomal proteins. It provides the scaffold for ribosome assembly but also prevents protein aggregation as part of the cellular stress response.

Mass Spectrometry and Machine Learning Reveal Determinants of Client Recognition by Antiamyloid Chaperones.

The assembly of proteins and peptides into amyloid fibrils is causally linked to serious disorders such as Alzheimer's disease. Multiple proteins have been shown to prevent amyloid formation in vitro and in vivo, ranging from highly specific chaperone-client pairs to completely nonspecific binding of aggregation-prone peptides. The underlying interactions remain elusive.

Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform.

Recombinant spider silk proteins (spidroins) have multiple potential applications in development of novel biomaterials, but their multimodal and aggregation-prone nature have complicated production and straightforward applications. Here, we report that recombinant miniature spidroins, and importantly also the N-terminal domain (NT) on its own, rapidly form self-supporting and transparent hydrogels at 37 °C. The gelation is caused by NT α-helix to β-sheet conversion and formation of amyloid-like fibrils, and fusion proteins composed of NT and green fluorescent protein or purine nucleoside phosphorylase form hydrogels with intact functions of the fusion moieties.

Structural Basis for Dityrosine-Mediated Inhibition of α-Synuclein Fibrillization.

α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized β-sheet structures that accumulate in plaques in brains of Parkinson's disease patients. Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation.

A "spindle and thread" mechanism unblocks p53 translation by modulating N-terminal disorder.

Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of "life on the edge of solubility.

Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry.

In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites.

Order and disorder-An integrative structure of the full-length human growth hormone receptor.

Because of its small size (70 kilodalton) and large content of structural disorder (>50%), the human growth hormone receptor (hGHR) falls between the cracks of conventional high-resolution structural biology methods. Here, we study the structure of the full-length hGHR in nanodiscs with small-angle x-ray scattering (SAXS) as the foundation. We develop an approach that combines SAXS, x-ray diffraction, and NMR spectroscopy data obtained on individual domains and integrate these through molecular dynamics simulations to interpret SAXS data on the full-length hGHR in nanodiscs.

Ion mobility-mass spectrometry shows stepwise protein unfolding under alkaline conditions.

Although native mass spectrometry is widely applied to monitor chemical or thermal protein denaturation, it is not clear to what extent it can inform about alkali-induced unfolding. Here, we probe the relationship between solution- and gas-phase structures of proteins under alkaline conditions. Native ion mobility-mass spectrometry reveals that globular proteins are destabilized rather than globally unfolded, which is supported by solution studies, providing detailed insights into alkali-induced unfolding events.

Molecular characteristics of porcine alpha-synuclein splicing variants.

Alpha-synuclein (α-syn) is a 140 amino acid, intrinsically disordered protein with a potential role in neurotransmitter vesicle release. The protein is natively unfolded under physiological conditions, and is expressed predominantly in neural tissue. α-syn is associated with neuropathological conditions in Parkinson's disease, where the protein misfolds into oligomers and fibrils resulting in aggregates in Lewy bodies.

Predicting the Shapes of Protein Complexes through Collision Cross Section Measurements and Database Searches.

In structural biology, collision cross sections (CCSs) from ion mobility mass spectrometry (IM-MS) measurements are routinely compared to computationally or experimentally derived protein structures. Here, we investigate whether CCS data can inform about the shape of a protein in the absence of specific reference structures. Analysis of the proteins in the CCS database shows that protein complexes with low apparent densities are structurally more diverse than those with a high apparent density.

Scratching the surface: native mass spectrometry of peripheral membrane protein complexes.

A growing number of integral membrane proteins have been shown to tune their activity by selectively interacting with specific lipids. The ability to regulate biological functions via lipid interactions extends to the diverse group of proteins that associate only peripherally with the lipid bilayer. However, the structural basis of these interactions remains challenging to study due to their transient and promiscuous nature.

A Mass-Spectrometry-Based Approach to Distinguish Annular and Specific Lipid Binding to Membrane Proteins.

Membrane proteins engage in a variety of contacts with their surrounding lipids, but distinguishing between specifically bound lipids, and non-specific, annular interactions is a challenging problem. Applying native mass spectrometry to three membrane protein complexes with different lipid-binding properties, we explore the ability of detergents to compete with lipids bound in different environments. We show that lipids in annular positions on the presenilin homologue protease are subject to constant exchange with detergent.

High intracellular stability of the spidroin N-terminal domain in spite of abundant amyloidogenic segments revealed by in-cell hydrogen/deuterium exchange mass spectrometry.

Proteins require an optimal balance of conformational flexibility and stability in their native environment to ensure their biological functions. A striking example is spidroins, spider silk proteins, which are stored at extremely high concentrations in soluble form, yet undergo amyloid-like aggregation during spinning. Here, we elucidate the stability of the highly soluble N-terminal domain (NT) of major ampullate spidroin 1 in the Escherichia coli cytosol as well as in inclusion bodies containing fibrillar aggregates.

Gas-Phase Collisions with Trimethylamine-N-Oxide Enable Activation-Controlled Protein Ion Charge Reduction.

Modulating protein ion charge is a useful tool for the study of protein folding and interactions by electrospray ionization mass spectrometry. Here, we investigate activation-dependent charge reduction of protein ions with the chemical chaperone trimethylamine-N-oxide (TMAO). Based on experiments carried out on proteins ranging from 4.

A strategy for the identification of protein architectures directly from ion mobility mass spectrometry data reveals stabilizing subunit interactions in light harvesting complexes.

Biotechnological applications of protein complexes require detailed information about their structure and composition, which can be challenging to obtain for proteins from natural sources. Prominent examples are the ring-shaped phycoerythrin (PE) and phycocyanin (PC) complexes isolated from the light-harvesting antennae of red algae and cyanobacteria. Despite their widespread use as fluorescent probes in biotechnology and medicine, the structures and interactions of their noncrystallizable central subunits are largely unknown.

Mechanistic Understanding of the Interactions between Nano-Objects with Different Surface Properties and α-Synuclein.

Aggregation of the natively unfolded protein α-synuclein (α-syn) is key to the development of Parkinson's disease (PD). Some nanoparticles (NPs) can inhibit this process and in turn be used for treatment of PD. Using simulation strategies, we show here that α-syn self-assembly is electrostatically driven.

Oleuropein derivatives from olive fruit extracts reduce α-synuclein fibrillation and oligomer toxicity.

Aggregation of α-synuclein (αSN) is implicated in neuronal degeneration in Parkinson's disease and has prompted searches for natural compounds inhibiting αSN aggregation and reducing its tendency to form toxic oligomers. Oil from the olive tree ( L.) represents the main source of fat in the Mediterranean diet and contains variable levels of phenolic compounds, many structurally related to the compound oleuropein.

Potent α-Synuclein Aggregation Inhibitors, Identified by High-Throughput Screening, Mainly Target the Monomeric State.

α-Synuclein (αSN) aggregation is central to the etiology of Parkinson's disease (PD). Large-scale screening of compounds to identify aggregation inhibitors is challenged by stochastic αSN aggregation and difficulties in detecting early-stage oligomers (αSOs). We developed a high-throughput screening assay combining SDS-stimulated αSN aggregation with FRET to reproducibly detect initial stages in αSN aggregation.